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Project
Proinflammatory and procoagulant response of human vascular endothelial cells in a model of endocarditis - contribution of tissue engineering.
Infective endocarditis (IE) still remains a clinical challenge in diagnosis and is associated with severe complications. </>Results of this thesis give further insight in the understanding of the EC activation process induced by S. aureus</>, </>one of the most important players causingacute valvular disease. </> </></>
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Fibronectin binding proteins of S. aureus</> were verified to mediate bacterial adhesion to ECs. We first described the role of FnBPs in inducing a variety of proinflammatory endothelial responses, resulting in leukocyte accumulation, cell damage and fibrin deposition. Using recombinant lactococci expressing staphylococcal</> surface molecules FnBPA or FnBPB, we found a very efficient inoculum-dependent pattern of EC infection in these strains. This coincided with EC activation, as we found a marked increase in surface expression of ICAM-1 and VCAM-1 as well as secretion of IL-8 and IL-6. The FnBPA-induced surface expression of ICAM-1 and VCAM-1 was similar to that found after stimulation with IL-1, a potent inducer of endothelial pro-inflammatory responses. </>
FnBPA-positive L. lactis</> also induced a prominent tissue factor-dependent endothelial coagulationresponse by elevated levels of TF-antigen and factor Xa activity. Furthermore, FnBPA mediated monocyte adhesion, which coincided with a significant increase in the amount of FXa formed. The ClfA-positive L. lactis</> strain showed a very low probability to infect ECs, which did not lead to EC activation. </>
From these results we conclude </>that S. aureus</> FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis</> organisms, indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.</>
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Further investigations addressed the c</>ontribution of </>the </>single or combined FnBPA subdomains (A, B, C or D) to the bacterial-endothelial interaction. </></>Using L. lactis</>, expressing single FnBPA (sub)domains or combinations thereof, we demonstrated that domains which mediate Fn-binding were necessary and sufficient to provoke thetypical EC responses. </>
FnBPA subdomains CD (aa 604-877) or A4+16 (aa 432-559) are involved in bacterial adhesion to ECs, which correlateswith the capacity of these fragments to elicit a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the IL-8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. I</>n addition, </></>our current data reveal that </>that only one single Fn binding modules is sufficient for maximal stimulation of endothelial proinflammatory and procoagulant responses. </></>
I</>n summary, this study gives further insight in the role S. aureus</> FnBPA plays in facilitating EC activation processes. Results identified the essential role of the tandemly repeated Fn-binding modules, expressed either alone or in combination, to both infect and activate ECs. </>
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In the final part of the thesis, our findings are transferred to an endocarditis model, which applies tissue engineering. Novel therapeutic strategies aim to minimize associated risk factors in valve replacement and to increase biocompatibility, by constructing autologous matrix-based tissue engineered heart valves. </>
The interactionof these matrices with bacteria is not known yet, but the potential role of these newly constructed heart valves, and especially their matrix, in the pathogenesis of endovascular infection seems important. Therefore, we compare the influence of a fibrin and collagen gel matrix on the bacterial-endothelial interaction, upon contact with S. aureus</>, S. epidermidis</> and S. sanguis. </></>
Results established a high capability of S. aureus</> to infect endothelial cells seeded on a fibrin and collagen matrix. In contrast, S. aureus</> bacteria adhered to the ECs seeded on the tissue culture plate showed significantly lower values. Infection of ECs with S. sanguis</> and S. epidermidis</> showed a similar, limited infection rate on these three surfaces. </>
The bacterial adhesion capacity was similar on the two gel matrices and resulted in endothelial proinflammatory and procoagulant activation. We found an importantendothelial activation with markedly expressed ICAM-1 and VCAM-1 valuesafter S. aureus</> infection, compared to a low increase of endothelialactivation after stimulation with S. sanguis</> and S. epidermidis</>.</>
Experimental results reveal no difference between the fibrin and collagen matrix but compared to results acquired on tissue culture platesa decreased monocyte mediated procoagulant activity was observed despite unchanged monocyte adhesion. </> </>Enhanced anticoagulant pathways, such as production of tissue factor pathway inhibitor (TFPI) or IL-10, might be involved. </></>
These investigations provide evidence for the importance of different matrices as inducers of pathways to evoke inflammation, tissue damage and fibrin deposition at infected endovascular sites. </></>
Collectively, this thesis gives further insight in the essential bacterial and cellular interactions in the early phases of the pathogenesis of S. aureus</> endocarditis. To develop anti-adhesive treatment strategies, the knowledge that FnBPA is identified as a central surface molecule and that at least one Fn-binding domain is necessary and sufficient to infect human ECs and, concomitantly, to launch aproinflammatory and procoagulant endothelial phenotype, is fundamental. </></>
</>
Fibronectin binding proteins of S. aureus</> were verified to mediate bacterial adhesion to ECs. We first described the role of FnBPs in inducing a variety of proinflammatory endothelial responses, resulting in leukocyte accumulation, cell damage and fibrin deposition. Using recombinant lactococci expressing staphylococcal</> surface molecules FnBPA or FnBPB, we found a very efficient inoculum-dependent pattern of EC infection in these strains. This coincided with EC activation, as we found a marked increase in surface expression of ICAM-1 and VCAM-1 as well as secretion of IL-8 and IL-6. The FnBPA-induced surface expression of ICAM-1 and VCAM-1 was similar to that found after stimulation with IL-1, a potent inducer of endothelial pro-inflammatory responses. </>
FnBPA-positive L. lactis</> also induced a prominent tissue factor-dependent endothelial coagulationresponse by elevated levels of TF-antigen and factor Xa activity. Furthermore, FnBPA mediated monocyte adhesion, which coincided with a significant increase in the amount of FXa formed. The ClfA-positive L. lactis</> strain showed a very low probability to infect ECs, which did not lead to EC activation. </>
From these results we conclude </>that S. aureus</> FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis</> organisms, indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.</>
</>
Further investigations addressed the c</>ontribution of </>the </>single or combined FnBPA subdomains (A, B, C or D) to the bacterial-endothelial interaction. </></>Using L. lactis</>, expressing single FnBPA (sub)domains or combinations thereof, we demonstrated that domains which mediate Fn-binding were necessary and sufficient to provoke thetypical EC responses. </>
FnBPA subdomains CD (aa 604-877) or A4+16 (aa 432-559) are involved in bacterial adhesion to ECs, which correlateswith the capacity of these fragments to elicit a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the IL-8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. I</>n addition, </></>our current data reveal that </>that only one single Fn binding modules is sufficient for maximal stimulation of endothelial proinflammatory and procoagulant responses. </></>
I</>n summary, this study gives further insight in the role S. aureus</> FnBPA plays in facilitating EC activation processes. Results identified the essential role of the tandemly repeated Fn-binding modules, expressed either alone or in combination, to both infect and activate ECs. </>
</>
In the final part of the thesis, our findings are transferred to an endocarditis model, which applies tissue engineering. Novel therapeutic strategies aim to minimize associated risk factors in valve replacement and to increase biocompatibility, by constructing autologous matrix-based tissue engineered heart valves. </>
The interactionof these matrices with bacteria is not known yet, but the potential role of these newly constructed heart valves, and especially their matrix, in the pathogenesis of endovascular infection seems important. Therefore, we compare the influence of a fibrin and collagen gel matrix on the bacterial-endothelial interaction, upon contact with S. aureus</>, S. epidermidis</> and S. sanguis. </></>
Results established a high capability of S. aureus</> to infect endothelial cells seeded on a fibrin and collagen matrix. In contrast, S. aureus</> bacteria adhered to the ECs seeded on the tissue culture plate showed significantly lower values. Infection of ECs with S. sanguis</> and S. epidermidis</> showed a similar, limited infection rate on these three surfaces. </>
The bacterial adhesion capacity was similar on the two gel matrices and resulted in endothelial proinflammatory and procoagulant activation. We found an importantendothelial activation with markedly expressed ICAM-1 and VCAM-1 valuesafter S. aureus</> infection, compared to a low increase of endothelialactivation after stimulation with S. sanguis</> and S. epidermidis</>.</>
Experimental results reveal no difference between the fibrin and collagen matrix but compared to results acquired on tissue culture platesa decreased monocyte mediated procoagulant activity was observed despite unchanged monocyte adhesion. </> </>Enhanced anticoagulant pathways, such as production of tissue factor pathway inhibitor (TFPI) or IL-10, might be involved. </></>
These investigations provide evidence for the importance of different matrices as inducers of pathways to evoke inflammation, tissue damage and fibrin deposition at infected endovascular sites. </></>
Collectively, this thesis gives further insight in the essential bacterial and cellular interactions in the early phases of the pathogenesis of S. aureus</> endocarditis. To develop anti-adhesive treatment strategies, the knowledge that FnBPA is identified as a central surface molecule and that at least one Fn-binding domain is necessary and sufficient to infect human ECs and, concomitantly, to launch aproinflammatory and procoagulant endothelial phenotype, is fundamental. </></>
Date:1 Apr 2009 → 24 Sep 2010
Keywords:Endothelial cells, Endocarditis
Disciplines:Cardiac and vascular medicine
Project type:PhD project